Bio-Rad Model 111 Mini IEF Cell - Manual (Page 10)

3.2 Stock Solutions for Polyacrylamide IEF Gels
Always use high quality distilled or deionized water to prepare stock solutions for
electrofocusing.
1. Monomer concentrate (T=25%, C=3%)
24.25% (w/v) acrylamide
0.75% (w/v) bis (N, N’-Methylene-bis-acrylamide)
Dissolve 24.25 g acrylamide and 0.75 g bis in water, bring to a final volume of 100 ml, and
filter through a 0.45 µm filter. Store protected from light at 4 °C. This solution may be stored
up to 1 month.
2. 0.1% (w/v) riboflavin-5'-phosphate (FMN)
50 mg riboflavin-5'-phosphate
50 ml water
This solution may be stored up to 1 month at 4 °C protected from light.
3. 10% (w/v) ammonium persulfate
100 mg ammonium persulfate
1 ml water
Prepare fresh daily. Make sure that the ammonium persulfate is completely dissolved before
using.
4. 25% glycerol (w/v)
Add 25 g glycerol to 50 ml H
2
O. Dilute to 100 ml with H
2
0.
5. TEMED (N,N’-tetramethylene-ethylenediamine)
Use TEMED neat from the bottle. Use only pure, distilled TEMED. Store cool, dry, and
protected from light.
3.3 Reagents for Polyacrylamide Electrofocusing Gels
The following volumes will produce sufficient reagent for two 125 x 65 x 0.4 mm gels:
Monomer-ampholyte solution
H
2
O 5.5 ml
Monomer concentrate (25% T, 3% C) 2.0 ml
25% (w/v) glycerol 2.0 ml
*Ampholyte 0.5 ml
Catalyst solutions
10% (w/v) ammonium persulfate 15 µl
0.1% (w/v) FMN 50 µl
TEMED (neat) 3 µl
*Volume required for 40% ampholyte solutions (Bio-Lyte 3/10, 4/6, 5/7, 6/8, 7/9 ampholytes).
For 20% ampholyte (Bio-Lyte 3/5, 8/10 ampholytes) add 1 ml ampholyte and reduce H2O
volume to 5.0 ml.
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